Angiotensin-(1-7) and angiotensin-(1-7) agonists for inhibition of cancer cell growth

ABSTRACT

The present invention describes the use of angiotensin-(1-7) peptide as an anti-cancer therapeutic. Thus, in one embodiment, the present invention comprises a composition to inhibit the growth of cancer cells in an individual comprising a pharmaceutically effective amount of an agonist for the angiotensin-(1-7) receptor to inhibit cancer cell growth or proliferation. Application of a pharmaceutically effective amount of angiotensin-(1-7) or angiotensin-(1-7) receptor agonist is associated with an increase in the expression of genes involved in tumor suppression, apoptosis, and/or cell cycle inhibition, and a decrease the expression of known oncogenes, protein kinases, and/or cell cycle progression genes. Cancers treated using the methods and compositions described herein include cancers having an angiotensin-(1-7) receptor, including, but not limited to, breast and lung cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 60/359,847, filed Feb. 27, 2002. The disclosure of U.S. ProvisionalApplication Ser. No. 60/359,847 is incorporated herein by reference inits entirety.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for thetreatment and prevention of cancer. More specifically, the presentinvention relates to the use of angiotensin-(1-7) or other agonists forthe angiotensin-(1-7) receptor as anticancer therapeutics.

BACKGROUND

Angiotensin-(1-7) [Ang-(1-7)] is an endogenous peptide hormone which isnormally present in the circulation at concentrations similar toangiotensin II (Ang II) and is primarily derived from angiotensin I (AngI) by tissue peptidases, including neprilysin, thimet oligopeptidase andprolyl endopeptidase (Ferrario, C. M. et al., Hypertension, 1997,30:535-541) and by angiotensin converting enzyme (ACE) 2 fromangiotensin II (Ang II) (Vickers, C., et al., J. Biol. Chem., 2002,277:14836-14843). In addition, Ang-(1-7) is a substrate for ACE(Chappell, M. C. et al. Hypertension, 1998, 31:362-367). ACE catalyzesthe conversion of angiotensin I (Ang I) to the biologically activepeptide angiotensin II [Ang II]. Treatment of patients or animals withACE inhibitors results in a significant elevation in the circulating andtissue levels of Ang II, as well as the N-terminal heptapeptide fragmentof Ang II, angiotensin-(1-7) (Campbell, M. C. et al., Hypertension,1993, 22:513-522; Kohara, K. et al., Hypertension, 1991,17:131-138Lawrence, A. C. et al., J. Hypertens., 1990, 8:715-724;andLuque, M. et al., J. Hypertens., 1996, 14:799-805). It has beensuggested that ACE inhibition not only elevates Ang-(1-7) by increasingAng I, the substrate for Ang-(1-7) production, but also by preventingAng-(1-7) conversion to the inactive fragment Ang-(1-5).

Although Ang-(1-7) was long-considered an inactive product of thedegradation of Ang II, studies showed that the heptapeptide producesunique physiological responses which are often opposite to those of thewell-recognized angiotensin peptide, Ang II (Ferrario, C. M. et al.,Hypertension, 1997, 30:535-541). Thus, Ang-(1-7) has been shown tostimulate vasopressin release from neuropeptidergic neurons (Schiavone,M. T. et al., Proc. Natl. Acad. Sci. USA, 1988, 85:4095-4098), increasethe release of certain neurotransmitters (Ambuhl, P. et al., Regul.Pept., 1992, 38:111-120), reduce blood pressure in hypertensive dogs andrats (Benter, I. F. et al., Am J. Physiol. Heart Circ. Physiol., 1995,269:H313-H319;and Nakamoto, H. et al., Hypertension, 1995, 25:796-802),and have biphasic effects on renal fluid absorption (DelliPizzi, A. etal., Br. J. Pharmacol., 1994, 111:1-3; DelliPizzi, A. et al.,Pharmacologist, 34, 1992; Garcia, N. H. and Garbin, J. L., J. Am. Soc.Nephrol., 1994, 5:1133-1138; Handa, R. K. et al., Am. J. Physiol., 1996,270:F141-F147;and Hilchey, S. D. and Bell-Quilley, C. P., Hypertension,1995, 25:1238-1244).

Besides its role in reducing blood pressure, Ang-(1-7) attenuatesvascular growth both in vitro and in vivo (Freeman, E. J. et al.,Hypertension, 1996, 28:104-108; Strawn, W. B. et al., Hypertension,1999, 33:207-211;and Tallant, E. A. et al., Hypertension, 1999,34:950-957). Also, hypertensive patients administered ACE inhibitorsshow a reduced risk of cancer, particularly lung and sex-specificcancers (Jick, H., et al., Lancet, 1997, 349:525-528; Lever, A. F. etal., Lancet, 1998, 352:179-184;and Pahor, M. et al., Am. J. Hypertens.,1996, 9:695-699).

What is needed in cancer prevention and therapeutics is a way to preventtumors from forming, or to inhibit the growth of tumors once formed.Also, what is needed are agents that act specifically at the tumor cell,thus minimizing non-specific and/or toxic side effects. Preferably, thechemotherapeutic agents will comprise ligands that target thechemotherapeutic agent to cancer cells with high efficacy to eitherreduce cellular signals that promote cell growth, or to increasecellular signals that promote cell death.

SUMMARY OF THE INVENTION

The present invention relates to the use of angiotensin-(1-7)[Ang-(1-7)] receptor agonists as anticancer therapeutics. Thus,embodiments of the present invention describes the use of agonists forthe Ang-(1-7) receptor, such as the Ang-(1-7) peptide and derivativesthereof, or agents which increase levels of plasma, tissue or cellularAng-(1-7), as compounds for prevention and treatment of cancer cellgrowth and proliferation.

Embodiments of the present invention recognize that Ang-(1-7) caninhibit tumor cell growth in vitro and in vivo. Preferably, cancerstreated by the method of the present invention comprise bladder cancer,breast cancer, brain cancer, colon cancer, endometrial cancer, head andneck cancer, leukemia, lymphoma, lung cancer, melanoma, liver cancer,rectal cancer, ovarian cancer, prostate cancer, bone cancer, pancreaticcancer, skin cancer, or renal cancer.

In one embodiment, the present invention comprises a composition forinhibition of cell growth or proliferation comprising a pharmaceuticallyeffective amount of an agonist for the angiotensin-(1-7) receptor in apharmaceutically acceptable carrier, wherein a pharmaceuticallyeffective amount of angiotensin-(1-7) receptor agonist comprises anamount which is sufficient to inhibit cell growth or proliferation.

In an embodiment, the present invention comprises a composition forinhibition of cancer cell growth or proliferation comprising apharmaceutically effective amount of an agonist for theangiotensin-(1-7) receptor in a pharmaceutically acceptable carrier,wherein a pharmaceutically effective amount of angiotensin-(1-7)receptor agonist inhibits growth or proliferation of the cancer cells.In an embodiment, the angiotensin-(1-7) receptor agonist comprisesangiotensin-(1-7) peptide having the sequence set forth in SEQ ID NO: 1.

Another embodiment of the present invention comprises a composition toinhibit the growth or proliferation of cancer cells in an individualcomprising a pharmaceutically effective amount of a compound whichprovides sufficient angiotensin-(1-7) receptor agonist to inhibit growthor proliferation of the cancer cells.

In one embodiment, the present invention comprises a method to inhibitcell growth or proliferation comprising application an agonist for theangiotensin-(1-7) receptor to the cells, wherein the cells have afunctional angiotensin-(1-7) receptor.

In another embodiment, the present invention comprises a method toinhibit the growth or proliferation of cancer cells in an individualcomprising application of a pharmaceutically effective amount of anagonist for the angiotensin-(1-7) receptor to the individual, wherein apharmaceutically effective amount comprises sufficient angiotensin-(1-7)receptor agonist to inhibit growth or proliferation of the cancer cells.

In yet another embodiment, the present invention comprises a method toinhibit the growth or proliferation of cancer cells in an individualcomprising application of a pharmaceutically effective amount of acompound which increases the efficacy or amount of circulating orcellular angiotensin-(1-7) receptor agonist.

In yet another embodiment, the present invention comprises a kit forinhibiting cancer cell growth and proliferation in an individualcomprising: (a) at least one container comprising a pharmaceuticallyeffective amount of a functional agonist for the angiotensin-(1-7)receptor; (b) a pharmaceutically acceptable carrier; and (c)instructions for use.

From the foregoing summary, it is apparent that an object of the presentinvention is to provide methods and compositions for the use ofangiotensin-(1-7) receptor agonists as anti-cancer therapeutics. Thereare, of course, additional features of the invention which will bedescribed hereinafter and which will form the subject matter of theclaims appended hereto. It is to be understood that the invention is notlimited in its application to the specific details as set forth in thefollowing description and figures. The invention is capable of otherembodiments and of being practiced or carried out in various ways.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the dose-dependent effect of angiotensin peptides on3H-thymidine incorporation into vascular smooth muscle cells (VSMCs) inaccordance with an embodiment of the present invention.

FIG. 2 shows inhibition of the 1 μM Ang-(1-7)(Asp-Arg-Val-Tyr-Ile-His-Pro) (SEQ ID NO: 1) mediated reduction (+A7) inserum-stimulated growth (FBS) by [Sar¹-Thr⁸]-Ang II (Sarthran)(Sar-Arg-Val-Tyr-Ile-His-Pro-Thr) (SEQ ID NO: 2) or [D-Ala⁷]-Ang-(1-7)(DAlaA7) (Asp-Arg-Val-Tyr-Ile-His-[D]Ala) (SEQ ID NO: 3) but not by AT₁(L158,809) or AT₂ (PD123177) receptor antagonists in accordance with anembodiment of the present invention.

FIG. 3 shows stained sections of: an uninjured rat carotid artery; asaline-treated injured carotid artery; and an injured carotid arterytreated with Ang-(1-7), in accordance with an embodiment of the presentinvention.

FIG. 4 shows morphometric analysis of intima and media of injured ratcarotid arteries and the media of uninjured rat carotid arteries inballoon catheter-injured rats infused with either saline or Ang-(1-7)(*P<0.05; n=8) in accordance with an embodiment of the presentinvention.

FIG. 5 shows that Ang-(1-7) causes a dose-dependent reduction inserum-stimulated ³H-thymidine incorporation into SK-LU-1, A549, andSK-MES-1 human lung cancer cells and ZR-75-1 human breast cancer cells(n=4-8, in triplicate) in accordance with an embodiment of the presentinvention.

FIG. 6 shows a time-dependent reduction in ³H-thymidine incorporationinto SK-LU-1, A549, and SK-MES-1 lung cancer cells and ZR-75-1 breastcancer cells in the presence of 100 nM Ang-(1-7) (n=3-4, in triplicate)in accordance with an embodiment of the present invention.

FIG. 7 shows that the Ang-(1-7)-stimulated reduction in ³H-thymidineincorporation into SK-LU-1 lung cancer cells is blocked by pretreatmentwith [D-Ala⁷]-Ang-(1-7) (DalaA7), but not by an AT₁ (Losartan) or AT₂(PD123177) receptor antagonist (n=3, in triplicate) in accordance withan embodiment of the present invention.

FIG. 8 shows that Ang-(1-7) (Asp-Arg-Val-Tyr-Ile-His-Pro) (SEQ ID NO:1), at 1 or 100 nM, reduced serum-stimulated ³H-thymidine incorporationinto SK-LU-1 lung cancer cells while Ang I(Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) (SEQ ID NO: 4), Ang-(2-8)(Arg-Val-Tyr-Ile-His-Pro-Phe) (SEQ ID NO: 5), Ang-(3-8)(Val-Tyr-Ile-His-Pro-Phe) (SEQ ID NO: 6), Ang-(3-7)(Val-Tyr-Ile-His-Pro) (SEQ ID NO: 7) and Ang II(Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) (SEQ ID NO: 8) were ineffective (n=3-9in triplicate; * indicates p<0.05) in accordance with an embodiment ofthe present invention.

FIG. 9 shows inhibition of breast cancer tumor growth by Ang-(1-7) inaccordance with an embodiment of the present invention. Tumor-bearingmice infused for 28 days with Ang-(1-7) (n=4) had a 40% reduction intumor size, while the tumors of saline-treated animals (n=3) doubled, ascompared to tumor volume prior to treatment.

FIG. 10 shows the effect of the cAMP-dependent protein kinase inhibitor(PKAI) Rp-cAMPS (10 μM) on the inhibition of serum-stimulated³H-thymidine incorporation by either 1 μM Ang-(1-7) or 5 μM carbacyclinin VSMCs in accordance with an embodiment of the present invention. Theresults are from VSMCs from 3 Sprague Dawley rats and each point was intriplicate.

FIG. 11 shows that increasing concentrations of Ang-(1-7) causes adose-dependent reduction in ERK1 and ERK 2 activities stimulated by 100nM Ang II (n=VSMCs from 7 different rat aortas; * denotes p<0.05), inaccordance with an embodiment of the present invention.

FIG. 12 shows that Ang-(1-7) causes a dose dependent reduction inserum-stimulated activation of ERK1 and ERK2 in SK-LU-1 lung cancercells in accordance with an embodiment of the present invention. Thedata is representative of experiments with SK-LU-1 cells of 3 differentpassage numbers.

FIG. 13 shows that Ang-(1-7) inhibits platelet-derived growth factor(PDGF) or epidermal growth factor (EGF)-stimulated ³H-thymidineincorporation into human ZR-75-1 breast cancer cells in accordance withan embodiment of the present invention.

FIG. 14 shows a histogram of SK-LU-1 cells treated with Ang-(1-7) inaccordance with an embodiment of the present invention. QuiescentSK-LU-1 lung cancer cells were stimulated with 1% FBS for 2 h in thepresence or absence of 100 nM Ang-(1-7). Radiolabeled CDNA, preparedfrom DNase-treated total RNA, was incubated with Human Cancer Atlas cDNAExpression Array (Clontech Laboratories).

FIG. 15 shows regulation of MEK 5 mRNA and protein by Ang-(1-7) inSK-LU-1 lung cancer cells in accordance with an embodiment of thepresent invention.

FIG. 16 shows that Ang-(1-7) stimulates apoptosis in mitogen-stimulatedSK-LU-1 lung cancer cells as evidenced by an increase in the caspase-3cleavage product poly(ADP-ribose) polymerase (PARP) as measured using anantibody specific to cleaved PARP in serum stimulated SK-LU-1 cellstreated for either 2, 4, or 8 h with 10 nM Ang-(1-7) in accordance withan embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes the use of angiotensin-(1-7) receptoragonists, such as angiotensin-(1-7) [Ang-(1-7)](Asp-Arg-Val-Tyr-Ile-His-Pro) (SEQ ID NO: 1) as anticancer therapeutics.Thus, embodiments of the present invention recognize that agonists ofthe Ang-(1-7) receptor can inhibit tumor cell growth in vitro and invivo.

In one embodiment, the present invention comprises a method to inhibitcell growth or proliferation comprising application of an agonist forthe angiotensin-(1-7) receptor to the cells wherein the cells have afunctional angiotensin-(1-7) receptor.

The receptor may be located in either the membrane or within thecellular compartments. Preferably, the cells comprise cancer cells. Morepreferably, the cancer comprises bladder cancer, breast cancer, braincancer, colon cancer, endometrial cancer, head and neck cancer,leukemia, lymphoma, lung cancer, melanoma, liver cancer, rectal cancer,ovarian cancer, prostate cancer, renal cancer, bone cancer, pancreaticcancer or skin cancer.

In an embodiment, the angiotensin-(1-7) receptor agonist comprisesangiotensin-(1-7) peptide having the sequence set forth in SEQ ID NO: 1.In an embodiment, the angiotensin-(1-7) receptor agonist is modified toincrease its chemical stability in vivo. In an alternate embodiment, theangiotensin-(1-7) receptor agonist comprises a fragment ofangiotensin-(1-7) or a functional equivalent of angiotensin-(1-7)comprising conservative amino acid substitutions, wherein conservativeamino acid substitutions are those substitutions which do notsignificantly effect the structure or function of the peptide. In yetanother embodiment, the angiotensin-(1-7) receptor agonist comprises anon-peptide agonist.

In yet another embodiment, the present invention comprises a method toinhibit the growth or proliferation of cancer cells in an individualcomprising application of a pharmaceutically effective amount of anagonist for the angiotensin-(1-7) receptor to the individual, wherein apharmaceutically effective amount comprises sufficient angiotensin-(1-7)receptor agonist to inhibit growth or proliferation of the cancer cells.Preferably, the individual is human.

Preferably, the cancer comprises cells having a functionalangiotensin-(1-7) receptor. The receptor may be on the cell membrane orintracellular. Also preferably, the cancer comprises bladder cancer,breast cancer, brain cancer, colon cancer, endometrial cancer, head andneck cancer, leukemia, lymphoma, lung cancer, melanoma, liver cancer,rectal cancer, ovarian cancer, prostate cancer, bone cancer, pancreaticcancer, skin cancer, or renal cancer.

In an embodiment, the angiotensin-(1-7) receptor agonist comprisesangiotensin-(1-7) peptide having the sequence set forth in SEQ ID NO: 1.In an embodiment, the angiotensin-(1-7) receptor agonist is modified toincrease its chemical stability in vivo. In an alternate embodiment,angiotensin-(1-7) receptor agonist comprises a fragment ofangiotensin-(1-7) or a functional equivalent of angiotensin-(1-7)comprising conservative amino acid substitutions, wherein conservativeamino acid substitutions are those substitutions which do notsignificantly effect the structure or function of the peptide. In yetanother embodiment, the angiotensin-(1-7) receptor agonist comprises anon-peptide agonist.

Also preferably, the method includes application of a compound whichincreases the efficacy or amount of circulating or cellularangiotensin-(1-7) agonist. For example, in an embodiment, the methodincludes application of a compound that increases angiotensin-(1-7)synthesis. Alternatively, the method includes application of a compoundthat decreases angiotensin-(1-7) agonist degradation. In an embodiment,the method includes application of a compound that is an antagonist ofother, non-angiotensin-(1-7) receptor subtypes, such as an anatagonistfor the AT₁ angiotensin receptor.

Also preferably, application of a pharmaceutically effective amount ofangiotensin-(1-7) receptor agonist in the individual increases cellularprostacyclins. Also preferably, application of a pharmaceuticallyeffective amount of angiotensin-(1-7) receptor agonist in the individualincreases cellular cAMP.

In an embodiment, application of a pharmaceutically effective amount ofangiotensin-(1-7) receptor agonist increases the expression of genesinvolved in tumor suppression, apoptosis, and/or cell cycle inhibitionin the cancer cells. Preferably, the genes showing increased expressioncomprise BAD, oncostatin M-specific beta subunit, PDCD2, EGF responsefactor 1, CASP4, RBQ-3, p16-INK, menin, checkpoint suppressor 1, BAK,apoptotic protease activating factor-1, SOCS-3, insulin-like growthfactor binding protein 2, B-myb or the fau tumor suppressor.

Alternatively, or additionally, application of a pharmaceuticallyeffective amount of angiotensin-(1-7) receptor agonist in the individualmay also decrease the levels of known oncogenes, protein kinases, and/orcell cycle progression genes in the cancer cells. Preferably, the genesshowing decreased expression comprise cell cycle entry regulator, ERK1,cell cycle progression 2 protein, p21/K-ras 2B oncogene, epithelial cellkinase, ser/thr kinase, MAP kinase kinase 5 (MEK5), beta catenin,tyrosine-protein kinase receptor tyro3 precursor, protein phosphatase 2AB56-alpha, cyclin-dependent kinase regulatory subunit (CDC28), celldivision protein kinase 6 (CDK6), c-myc oncogene, ERBB-3 receptorprotein tyrosine kinase, A-kinase anchoring protein, or rho C.

In an embodiment, there is a discrete dosage range of angiotensin-(1-7)receptor agonist which is effective in inhibiting tumor cell growth.Preferably, the dose of angiotensin-(1-7) receptor agonist results in alocal concentration of angiotensin-(1-7) receptor agonist at the cancerwhich ranges from 0.005 nM to 10 μM. More preferably, the dose ofangiotensin-(1-7) receptor agonist results in a local concentration ofangiotensin-(1-7) receptor agonist at the cancer which ranges from 0.05nM to 1 μM. Even more preferably, the dose of angiotensin-(1-7) receptoragonist results in a local concentration of angiotensin-(1-7) orangiotensin-(1-7) receptor agonist at the cancer which ranges from 1 nMto 100 nM.

In yet another aspect, the present invention comprises a method toinhibit the growth or proliferation of cancer cells in an individualcomprising application of a pharmaceutically effective amount of acompound to the individual which increases the efficacy or amount ofcirculating or cellular angiotensin-(1-7) agonist.

In an embodiment, the compound which increases the efficacy or amount ofcellular angiotensin-(1-7) agonist increases angiotensin-(1-7)synthesis. In another embodiment, the compound which increases theefficacy or amount of cellular angiotensin-(1-7) agonist may decreaseangiotensin-(1-7) agonist degradation, metabolism or clearance. In yetanother embodiment, the compound which increases the efficacy or amountof cellular angiotensin-(1-7) agonist comprises an angiotensin AT₁receptor antagonist. Preferably, the cancer treated by the method of thepresent invention comprises bladder cancer, breast cancer, brain cancer,colon cancer, endometrial cancer, head and neck cancer, leukemia,lymphoma, lung cancer, melanoma, liver cancer, rectal cancer, ovariancancer, prostate cancer, bone cancer, pancreatic cancer, skin cancer, orrenal cancer.

In another aspect, the present invention comprises a composition forinhibition of cell growth or proliferation comprising a pharmaceuticallyeffective amount of an agonist for the angiotensin-(1-7) receptor in apharmaceutically acceptable carrier, wherein a pharmaceuticallyeffective amount of angiotensin-(1-7) receptor agonist comprises anamount which is sufficient to inhibit cell growth or proliferation.Preferably, the cells comprise cancer cells. More preferably, the cancercomprises bladder cancer, breast cancer, brain cancer, colon cancer,endometrial cancer, head and neck cancer, leukemia, lymphoma, lungcancer, melanoma, liver cancer, rectal cancer, ovarian cancer, prostatecancer, renal cancer, bone cancer, pancreatic cancer or skin cancer.

In another aspect, the present invention comprises a composition forinhibition of cancer cell growth or proliferation comprising apharmaceutically effective amount of an agonist for theangiotensin-(1-7) receptor in a pharmaceutically acceptable carrier,wherein a pharmaceutically effective amount of angiotensin-(1-7)receptor agonist comprises an amount which is sufficient to inhibitcancer cell growth and/or proliferation.

Preferably, the cancer comprises cells having a functionalangiotensin-(1-7) receptor. The receptor may be located on the cellmembrane or intracellular. For example, the cancer may comprise cellswhose functional angiotensin-(1-7) receptor signals or secretes achemical or protein that inhibits cancer cell growth. Also, in anembodiment, the cancer is in a human subject. Also preferably, thecancer comprises bladder cancer, breast cancer, brain cancer, coloncancer, endometrial cancer, head and neck cancer, leukemia, lymphoma,lung cancer, melanoma, liver cancer, rectal cancer, ovarian cancer,prostate cancer, renal cancer, bone cancer, pancreatic cancer, or skincancer.

In an embodiment, the angiotensin-(1-7) receptor agonist of thecomposition comprises angiotensin-(1-7) peptide having the sequence setforth in SEQ ID NO: 1. In an embodiment, the angiotensin-(1-7) receptoragonist of the composition is modified to increase its chemicalstability in vivo. In an alternate embodiment, the angiotensin-(1-7)receptor agonist comprises a fragment of angiotensin-(1-7) or afunctional equivalent of angiotensin-(1-7) comprising conservative aminoacid substitutions, wherein conservative amino acid substitutions arethose substitutions which do not significantly effect the structure orfunction of the peptide. In yet another embodiment, theangiotensin-(1-7) receptor agonist comprises a non-peptide agonist.

Also preferably, the composition includes a compound which increases theefficacy or amount of circulating or cellular angiotensin-(1-7) agonist.In an embodiment, the compound which increases the efficacy or amount ofangiotensin-(1-7) agonist increases angiotensin-(1-7) synthesis. Inanother embodiment, the compound which increases the efficacy or amountof angiotensin-(1-7) agonist may decrease angiotensin-(1-7) degradation,metabolism or clearance. In yet another embodiment, the compound whichincreases the efficacy or amount of angiotensin-(1-7) agonist comprisesa non-Ang-(1-7) angiotensin receptor antagonist, such an antagonist ofthe AT₁ angiotensin receptor.

In an embodiment, a pharmaceutically effective amount ofangiotensin-(1-7) receptor agonist increases cellular prostacyclins. Inan embodiment, a pharmaceutically effective amount of angiotensin-(1-7)receptor agonist increases cellular cAMP.

Also, in an embodiment, a pharmaceutically effective amount ofangiotensin-(1-7) receptor agonist increases the expression of genesinvolved in tumor suppression, apoptosis, and/or cell cycle inhibition.Preferably, the genes showing increased expression comprise BAD,oncostatin M-specific beta subunit, PDCD2, EGF response factor 1, CASP4,RBQ-3, p16-INK, menin, checkpoint suppressor 1, BAK, apoptotic proteaseactivating factor-1, SOCS-3, insulin-like growth factor binding protein2, B-myb or the fau tumor suppressor.

Alternatively, or additionally, a pharmaceutically effective amount ofangiotensin-(1-7) receptor agonist may decrease the levels of knownoncogenes, protein kinases, and/or cell cycle progression genes in thecancer. Preferably, the genes showing decreased expression comprise cellcycle entry regulator, ERK1, cell cycle progression 2 protein, p21/K-ras2B oncogene, epithelial cell kinase, ser/thr kinase, MAP kinase kinase 5(MEK5), beta catenin, tyrosine-protein kinase receptor tyro3 precursor,protein phosphatase 2A B56-alpha, cyclin-dependent kinase regulatorysubunit (CDC28), cell division protein kinase 6 (CDK6), c-myc oncogene,ERBB-3 receptor protein tyrosine kinase, A-kinase anchoring protein, orrho C.

In an embodiment, there is a discrete dosage range of angiotensin-(1-7)receptor agonist which is effective in inhibiting tumor cell growth.Preferably, the dose of angiotensin-(1-7) receptor agonist results in alocal concentration of angiotensin-(1-7) receptor agonist at the cancerwhich ranges from 0.005 nM to 10 μM. More preferably, the dose ofangiotensin-(1-7) receptor agonist results in a local concentration ofangiotensin-(1-7) receptor agonist at the cancer which ranges from 0.05nM to 1 μM. Even more preferably, the dose of angiotensin-(1-7) receptoragonist results in a local concentration of angiotensin-(1-7) receptoragonist at the cancer which ranges from 1 nM to 100 nM.

In another aspect, the present invention comprises a composition toinhibit the growth of cancer cells in an individual comprising apharmaceutically effective amount of a compound which increases theefficacy or amount of circulating or cellular angiotensin-(1-7) agonistin a pharmaceutical carrier, wherein a pharmaceutically effective amountprovides endogenous levels of angiotensin-(1-7) receptor agonist whichis sufficient to inhibit cancer cell growth or proliferation. Forexample, in an embodiment, the method includes application of a compoundthat increases angiotensin-(1-7) synthesis. In another embodiment, thecompound which increases the efficacy or amount of cellularangiotensin-(1-7) agonist may decrease angiotensin-(1-7) agonistdegradation, metabolism or clearance. In yet another embodiment, thecompound which increases the efficacy or amount of cellularangiotensin-(1-7) agonist comprises a non-Ang-(1-7) angiotensin receptorantagonist, such as an antagonist of the AT₁ angiotensin receptor.

For example, such compounds may include ACE inhibitors, or anypharmaceutical that blocks either the AT₁ angiotensin II receptor. Suchcompounds act to cause an increase in Ang-(1-7) and thereby, cancontribute to Ang-(1-7) mediated inhibition of cancer growth. In anembodiment, the compounds comprise angiotensin receptor blockers.

Preferably, the cancer treated by the method of the present inventioncomprises bladder cancer, breast cancer, brain cancer, colon cancer,endometrial cancer, head and neck cancer, leukemia, lymphoma, lungcancer, melanoma, liver cancer, rectal cancer, ovarian cancer, prostatecancer, bone cancer, pancreatic cancer, skin cancer, or renal cancer.

In yet another aspect, the present invention comprises a kit forinhibiting cancer cell growth in an individual comprising: (a) at leastone container comprising a pharmaceutically effective amount of afunctional agonist for the angiotensin-(1-7) receptor, wherein apharmaceutically effective amount comprises an amount ofangiotensin-(1-7) receptor agonist which is sufficient to inhibit cancercell growth or proliferation; (b) a pharmaceutically acceptable carrier;and (c) instructions for use.

Preferably, the cancer cells comprise a functional angiotensin-(1-7)receptor. The receptor may be located on the cell membrane, orintracellular. In an embodiment, the cancer comprises bladder cancer,breast cancer, brain cancer, colon cancer, endometrial cancer, head andneck cancer, leukemia, lymphoma, lung cancer, melanoma, liver cancer,rectal cancer, ovarian cancer, prostate cancer, bone cancer, pancreaticcancer, skin cancer, or renal cancer.

In an embodiment, the angiotensin-(1-7) receptor agonist comprisesangiotensin-(1-7) peptide having the sequence set forth in SEQ ID NO: 1.Preferably, the angiotensin-(1-7) receptor agonist used in the kit ismodified to increase its chemical stability in vivo. In an embodiment,the angiotensin-(1-7) receptor agonist comprises a fragment ofangiotensin-(1-7) or a functional equivalent of angiotensin-(1-7)comprising conservative amino acid substitutions. Alternatively, theangiotensin-(1-7) receptor agonist, may comprise a non-peptide agonist.

Also preferably, the kit includes a compound which increases theefficacy or amount of cellular angiotensin-(1-7) agonist in the cells.In an embodiment, the compound which increases the efficacy or amount ofcellular angiotensin-(1-7) increases angiotensin-(1-7) synthesis. Inanother embodiment, the compound which increases the efficacy or amountof cellular angiotensin-(1-7) agonist may decrease angiotensin-(1-7)agonist degradation, metabolism or clearance. In yet another embodiment,the compound which increases the efficacy or amount of cellularangiotensin-(1-7) agonist comprises a non-Ang-(1-7) angiotensin receptorantagonist, such as an antagonist of the AT₁ angiotensin receptor.

Angiotensin-(1-7) is a Physiological Mediator of Cell Growth

Studies indicate that the angiotensin peptides may be associated with avariety of cellular activities. Still, angiotensin-(1-7) has long beenconsidered an inactive product of AngII degradation. Only a few studieshave implicated angiotensin peptides as potentially having a role in theregulation of cell growth and/or cancer. For example, in two studies,patients receiving ACE inhibitors were found to have reduced relativerisk (0.73 and 0.79) of cancer (Jick, H. et al., Lancet, 1997,349:525-528;and Pahor, M. et al., Am. J. Hypertens., 1996, 9:695-699).These reductions in risk were not, however, statistically significant.In a retrospective study of 5207 patients in Scotland, the relativerisks of incident and fatal cancer among the 1559 patients treated withACE inhibitors were reduced, to 0.72 and 0.65, respectively, with therelative risk lowest in patients with lung or sex-specific cancer(Lever, A. F. et al., Lancet, 1998, 352:179-184). Other studiessuggested that treatment with ACE inhibitors may attenuate the growth ofpreneoplastic liver cells (Volpert, J. J. et al., J. Clin. Invest.,1996, 98:671-679) and renal cell carcinoma (Hii, S. I. et al., BritishJournal of Cancer, 1998, 77:880-883). While these studies suggest a rolefor ACE inhibitors in reducing cancer risk, there is no indication as tothe mechanism by which a lower risk of cancer may have occurred, or thatAng-(1-7) played a role.

The present invention describes the use of angiotensin-(1-7) [Ang-(1-7)]peptide and other Ang-(1-7) receptor agonists to inhibit cancer growth.Thus, in an embodiment, the present invention recognizes that Ang-(1-7)peptide (Asp-Arg-Val-Tyr-Ile-His-Pro) (SEQ ID NO: 1) binds to a specificreceptor present on tumor cells to affect second messengers associatedwith regulation of cell growth and proliferation. In an embodiment, thepresent invention describes that Ang-(1-7) binds to specific receptorsto invoke a decrease in the expression of genes associated with cellgrowth and proliferation and to invoke an increase in expression ofgenes associated with suppression of cell proliferation and/or apoptosisand cell death.

Thus, in one embodiment, the present invention comprises a method toinhibit cell proliferation comprising application of angiotensin-(1-7)[Ang-(1-7)] or other agonists for the angiotensin-(1-7) receptor to thecells of interest. In another embodiment, the present inventioncomprises a method to inhibit cell proliferation comprising applicationof a compound which increases the efficacy or amount of cellularangiotensin-(1-7) to cells comprising the Ang-(1-7) receptor. Forexample, and referring now to FIGS. 1-4, angiotensin-(1-7) inhibitsvascular smooth muscle cell (VSMC) growth both in vitro (FIGS. 1 and 2),and in vivo (FIGS. 3 and 4), suggesting that Ang-(1-7) may act as anendogenous regulator of cell growth.

Thus, FIG. 1 shows the dose-dependent effect of angiotensin peptides on3H-thymidine incorporation into vascular smooth muscle cells (VSMCs). Itcan be seen that Ang-(1-7) inhibits thymidine incorporation into DNA ina dose-dependent manner with an effective concentration for 50%inhibition (IC50) of about 115 nM. FIG. 2 shows that the ability ofAng-(1-7) to inhibit thymidine uptake is receptor mediated. Thus, asshown in FIG. 2, the 1 μM Ang-(1-7)-mediated reduction (+A7) inserum-stimulated growth (FBS) is inhibited by [Sar¹-Thr⁸]-Ang II(Sarthran) or [D-Ala⁷]-Ang-(1-7) (DalaA7) (which bind to the Ang-(1-7)receptor) but not by AT₁ (L158,809) or AT₂ (PD123177) receptorantagonists.

FIG. 3 shows stained sections of an uninjured rat carotid artery, asaline-treated injured carotid artery, and an injured corotid arterytreated with Ang-(1-7). Thus, in an embodiment, Ang-(1-7) reverses thecellular proliferation seen upon vascular injury. Morphometric analysisof carotid artery cross-sections indicates that Ang-(1-7) infusionsignificantly reduces the neointimal area compared to rats infused withsaline but has no effect on the medial area of the injured or thecontralateral uninjured artery as compared to saline controls (FIGS. 3and 4). Thus, Ang-(1-7) inhibits vascular growth in vivo and may preventvascular re-stenosis mediated by the proliferative response of smoothmuscle cells in blood vessels. For example, vascular re-stenosis is acomplication seen when vascular stents are used to prevent vesselocclusion in response to angioplasty and similar procedures.

The Effects of Ang-(1-7) Are Mediated by a Specific Receptor

In an embodiment, the effect of Ang-(1-7) on cell growth and/orproliferation is receptor mediated. Ang-(1-7) is a poor competitor atthe prototypical AT₁ angiotensin receptor in VSMC (Jaiswal, N. et al.,Hypertension, 1993, 21:900-905;and Jaiswal, N. et al., J. Pharmacol.Exp. Ther., 1993, 265:664-673) or the AT₂ angiotensin receptor(Chappell, M. C. et al., Peptides, 1995, 16:741-747;and Tallant, E. A.et al., Hypertension, 1991, 17:1135-1143). Thus, Ang-(1-7) displays IC₅₀levels in the micromolar range at the AT₁ or AT₂ angiotensin receptor(Tallant, E. A. et al., Hypertension, 1999, 34:950-957).

As described herein, IC₅₀ is the concentration of an agent whichprovides 50% of the total inhibition detected for a biological effect ofinterest, as for example, 50% inhibition of receptor binding or 50%inhibition of ³H-thymidine uptake.

Angiotensin receptors are pharmacologically defined by their selectivityfor the prototypical ligand losartan and similar antagonists such as L-158,809, while AT₂ receptors show selectivity for the antagonist PD123177 or PD 123319 (de Gasparo et al., 1995). Ang II, by stimulation ofAT₁ receptors, is a potent vasoconstrictor and stimulates thirst andaldosterone release. Inhibition of its production or effect using ACEinhibitors or AT₁ receptor antagonists reduces mean arterial pressure(Tallant, E. A. and Ferrario, C. M. Exp. Opin. Invest. Drugs 1996,5:1201-1214). In contrast, activation of AT₂ receptors by Ang II isassociated with vasodilation and reduced cell growth (Carey R. M. etal., Am. J. Hypertens. 2001, 6:98-1-2).

[D-Ala⁷]-Ang-(1-7), a modified form of Ang-(1-7), selectively blocksresponses to Ang-(1-7). [D-Ala⁷]-Ang-(1-7) is a poor competitor at theAT₁ or AT₂ receptor, and does not block pressor or contractile responsesto Ang II (Britto, R. R. et al., Hypertension, 1997, 30:549-556; Fontes,M. A. P. et al., (Brain Res., 1994, 665:175-180Oliveira, D. R. et al.,Hypertension, 27:1998, 1284-1290;and Santos, R. A. S. et al., Brain Res.Bull., 1994, 35:293-298). Thus, an Ang-(1-7) binding site on bovineaortic endothelial cells [BAEC] which was competed for by[Sar¹-Ile⁸]-AngII and [D-Ala⁷]-Ang-(1-7) but not by losartan or PD123319has been identified (Tallant, E. A. et al., Hypertension, 1996,29:388-393;and Heitsch, H. et al., Hypertension, 2001, 37:72-76). Asimilar ¹²⁵I-Ang-(1-7) binding site, sensitive to Ang-(1-7) and[D-Ala⁷]-Ang-(1-7), found in the endothelium of canine coronary arteryrings (Ferrario, C. M. et al., Hypertension, 1997, 30:535-541),consistent with functional effects of Ang-(1-7) in canine and porcinecoronary arteries (Brosnihan, K. R. and Ferrario, C. M., Hypertension,1996, 27:523-528;and Porsti, I. et al., Br. J. Pharmacol., 1994,111:652-654). As described herein, a similar binding site for Ang-(1-7)has been identified on VSMCs (Iyer, S.N., et al., J. Cardiovasc.Pharmacol., 2000, 36:109-117).

Thus, there is a specific angiotensin-(1-7) [Ang-(1-7)] receptor, thatis sensitive to [Sar¹-Thr⁸]-Ang II or [D-Ala⁷]-Ang-(1-7) but not tolosartan or PD123319. In an embodiment, the action of Ang-(1-7) toinhibit cell growth and/or cell proliferation comprises an interactionwith a specific receptor for Ang-(1-7). As described herein, thisangiotensin-(1-7) receptor may be referred to as the AT₍₁₋₇₎ receptor,in accordance with the guidelines established by the International Unionof Pharmacology Nomenclature Subcommittee for Angiotensin Receptors(Bumpus, F. M. et al., Hypertension, 1991, 17:720-721;and De Gasparo, M.et al., Hypertension, 1995, 25:924-927). The AT₍₁₋₇₎ receptor (orAng-(1-7) receptor) is defined by its sensitivity to Ang-(1-7), itsantagonism by [Sar¹-Thr⁸]-Ang II and [D-Ala⁷]-Ang-(1-7), and its lack ofresponse to losartan or PD123319, either functionally, or in competitionfor binding.

For example, and referring again to FIG. 2, inhibition ofmitogen-stimulated VSMC growth by Ang-(1-7) is not prevented by the AT₁antagonist L158,809 or the AT₂ antagonist PD 123319. However,[Sar¹-Thr⁸]-Ang II or the Ang-(1-7) antagonist [D-Ala⁷]-Ang-(1-7)effectively blocks growth inhibition of VSMCs by Ang-(1-7). Also, in anembodiment, the inhibition of serum-stimulated growth in cancer cells isattenuated by the selective Ang-(1-7) antagonist [D-Ala⁷]-Ang-(1-7), butnot by an AT₁ or AT₂ receptor antagonists (see FIG. 7, and discussionbelow).

In an embodiment, agonists other than Ang-(1-7) for the Ang-(1-7)receptor may be used in the methods of the present invention. In yetanother embodiment, non-peptide agonists such as those described in U.S.Pat. Nos. 6,429,222 and 6,235,766 (incorporated in their entireties byreference hererein) may be employed.

In an embodiment, the angiotensin-(1-7) or other angiotensin-(1-7)receptor agonist is chemically modified to increase its stability invivo. For example, to increase stability, the peptide may be modified atseveral positions to protect against aminopeptidase and endopeptidasehydrolysis. For aminopeptidase protection, the amino (N) terminus of thepeptide may be modified by substituting sarcosine for aspartic acid(Asp) or acetylated aspartic acid for aspartic acid. To protect againstendopeptidase attack, primarily ACE hydrolysis which occurs at theIle⁵-His⁶ bond of Ang-(1-7), D-isoleucine and D-histidine may besubstituted for isoleucine at position 5 (Ile⁵) and histidine atposition 6 (His⁶), respectively, of the peptide. Additionally, a reducedor methyline isostere bond may be introduced between Ile⁵ and His⁶.

In yet another embodiment, the angiotensin-(1-7) or angiotensin-(1-7)receptor agonist comprises a fragment of angiotensin-(1-7) or afunctional equivalent of angiotensin-(1-7) having conservative aminoacid substitutions, wherein conservative amino acid substitutions aredefined to be those amino acid substitutions which do not affect theapparent structure, or inhibit the function, of the peptide.

Angiotensin-(1-7) Inhibits Cancer Cell Growth and Proliferation

In an embodiment, the present invention describes the use of agonistsfor the Ang-(1-7) receptor to inhibit growth and proliferation of cancercells. Preferably, the Ang-(1-7) agonist may be used for inhibition ofbreast or lung cancer tumor growth (FIGS. 5-9). The inhibition of tumorgrowth by Ang-(1-7) seen in vitro (FIGS. 5-8) is also seen in vivo (FIG.9) indicating that Ang-(1-7) is effective for tumor reduction in vivo.

Thus, Ang-(1-7) inhibits growth of human lung cancer cells (SK-LU-1,A549, SK-MES-1) and breast cancer cells (ZR-75-1), in a dose-dependentmanner (FIG. 5). In an embodiment, the dose of Ang-(1-7) required forinhibition of cancer cells comprises levels of angiotensin-(1-7) usedpharmacologically in animals or humans. Also preferably, the dose ofangiotensin-(1-7) receptor agonist results in a local concentration ofangiotensin-(1-7) agonist at the tumor which ranges from 0.0005 nM to 10μM, and more preferably, from 0.05 nM to 1 μM, or even more preferably,from 1 nM to 100 nM (FIG. 5).

Thus, as shown in FIG. 5, Ang-(1-7) reduced tumor cell growth with anIC₅₀ of 0.05 nM for SK-LU-1 lung cancer cells, an IC₅₀ of 0.11 nM forA549 lung cancer cells, an IC₅₀ of 0.4 nM for SK-MES-1 lung cancercells, and an IC₅₀ of 0.02 nM for ZR-75-1 breast cancer cells Theseconcentrations of Ang-(1-7) are well within the range of Ang-(1-7) dosesused pharmacologically in animals or humans.

In an embodiment, the ability of Ang-(1-7) to inhibit tumor growth is afunction of cell division and the length of the cell cycle. For example,the incorporation of ³H-thymidine into SK-LU-1, A549, and SK-MES-1 lungcancer cells and ZR-75-1 breast cancer cells stimulated to grow by theinclusion of 1% FBS is progressively reduced by daily addition of 100 nMAng-(1-7) (FIG. 6). Thus, application of Ang-(1-7) may be hourly, daily,or over the course of weeks (FIG. 6).

Also, in an embodiment, the inhibition of serum-stimulated growth incancer cells is attenuated by the selective Ang-(1-7) antagonist[D-Ala⁷]-Ang-(1-7), but not by AT₁ or AT₂ receptor antagonists (FIG. 7).Thus, inhibition of the serum-stimulated growth of SK-LU-1 human lungcancer cells by Ang-(1-7) is blocked by the Ang-(1-7) selectiveantagonist [D-Ala⁷]-Ang-(1-7), while neither ATI nor AT₂ angiotensinreceptor antagonists, Losartan and PD123177, respectively, are effective(FIG. 7). This suggests that the anti-proliferative effect of Ang-(1-7)in cancer cells is mediated by a novel AT₍₁₋₇₎ receptor.

Also, the effects of Ang-(1-7) on cell growth and proliferation arespecific to Ang-(1-7), and are not exhibited by other angiotensinpeptides. Thus, neither Ang I, Ang-(2-8) or Ang III, Ang-(3-8) or AngIV, Ang-(3-7), nor Ang II mimicked the growth inhibitor effects ofAng-(1-7), as shown in FIG. 8. These results suggest that theanti-proliferative effect of Ang-(1-7) is mediated by a novel Ang-(1-7)receptor and may represent a new therapeutic treatment for thesecancers.

The effects of Ang-(1-7) on tumor growth are also seen in vivo. In amouse model using athymic mice injected with breast cancer cells, tumorgrowth is dramatically reduced upon infusion of Ang-(1-7) (24 μg/kg/hr)for 28 days. As shown in FIG. 9, an approximate 40% reduction in tumorvolume is observed in mice treated with Ang-(1-7) for 4 weeks, while thetumor size doubles in the saline-treated animals, as compared to tumorsize prior to treatment. These results show that Ang-(1-7) inhibitsbreast tumor growth in vivo and that Ang-(1-7) is an effectivetherapeutic agent in vivo.

Angiotensin-(1-7) and Intracellular Signaling

One major response to treatment of cells, tissues or whole animals withAng-(1-7) is the production of prostaglandins. Thus, in an embodiment,application of a pharmaceutically effective amount of angiotensin-(1-7)or angiotensin-(1-7) receptor agonist increases prostaglandins,prostacyclins and/or intracellular cAMP.

Ang-(1-7) induces prostaglandin release from astrocytes, porcine EC, andrat, porcine and rabbit VSMC (Jaiswal, N. et al., Hypertension, 1992,19:II-49-55; Jaiswal, N. et al., Am. J. Physiol. Regul. Integr. Comp.Physiol., 1991, 260:R1000-R1006; Jaiswal, N. et al., Hypertension, 1993,21:900-905; Jaiswal, N. et al., Hypertension, 1991, 17:1115-1120;Jaiswal, N. et al., J. Pharmacol. Exp. Ther., 1993, 264:664-673;Muthalif, M. M. et al., J. Pharmacol. Exp. Ther., 1998, 284:388-398;andTallant, E. A. et al., Hypertension, 1991, 18:32-39). For example, thevasodilator response to Ang-(1-7) and the depressor component of theresponse to Ang-(1-7) are reduced by prior treatment with thecyclooxygenase inhibitor indomethacin, indicating that these responseswere mediated by prostaglandins (Benter, I. F. et al., Peptides, 1993,14, 679-684; Meng, W. and Busija, D. W., Stroke, 1993, 24:2041-2045;andIyer, S. N. et al., J. Cardiovasc. Pharmacol., 2000, 36:109-117).

Prostacyclin (PGI₂) is a type of prostaglandin. Prostacylin is a potentvasodilator and reduces vascular growth via production of cAMP.Prostacyclin is produced by the cyclooxygenase-mediated conversion ofarachidonic acid into PGG₂/PGH₂, which is subsequently processed byprostacyclin synthase into prostacyclin. Interestingly, thecyclooxygenase inhibitor indomethacin effectively blocks the growthinhibition mediated by Ang-(1-7).

The addition of prostacyclin (or stable analogs of prostacyclin such ascarbacyclin) to VSMCs activates adenylate cyclase resulting in anelevation in the cellular levels of cAMP. Ang-(1-7), at a concentrationof 1 μM, causes a significant increase in the cellular levels of cAMP,to 131.9±9.7% of basal (n=3, p<0.05), in the presence of 1 mMisobutylmethyl xanthine (IBMX), a cyclic nucleotide phosphodiesteraseinhibitor. cAMP activates a cAMP-dependent protein kinase, proteinkinase A. As shown in FIG. 10, the reduction in serum-stimulated³H-thymidine incorporation by Ang-(1-7) or carbacyclin was completelyblocked by pretreatment with the protein kinase A inhibitor (PKAI)Rp-adenosine-3′,5′-cyclic monphospho-phorothioate triethylamine salt(Rp-cAMPS). These results suggest that Ang-(1-7) is directly coupled tothe Gs protein to activate adenylate cyclase and elevate cellular cAMPproduction. Alternatively, Ang-(1-7) may stimulate the production ofprostacyclin which binds to prostacyclin receptors coupled to adenylatecyclase and the synthesis of cAMP. Thus, in an embodiment, Ang-(1-7)causes an increase in the cellular levels of cAMP (directly or viaprostacylin) which stimulates the cAMP-dependent protein kinase toinhibit growth.

Alternatively and/or additionally, Ang-(1-7) may inhibit cell growth bypreventing the phosphorylation and activation of MAP kinases in responseto mitogen stimulation. Compounds that increase the intracellularconcentration of cAMP have been shown to reduce MAP kinase activity inVSMCs and fibroblasts and thereby inhibit mitogen-stimulated growth inVSMCs (Cook, S. J. and McCormick, F., Science, 1993, 262:1069-1072;andWu, J. et al., Science, 1993, 262;1065-1068). In addition, classicgrowth factors, such as PDGF, epidermal growth factor, and basicfibroblast growth factor stimulate VSMC growth in vitro and in vivo.Growth stimulation by these mitogens as well as by Ang II is mediated,at least in part, through activation of MAP kinases to induce earlyresponse genes and increase transcription.

The activity of the MAP kinases ERK1 and ERK2 in VSMCs can be measuredusing phospho-specific antibodies that only recognize the activatedprotein kinases. As shown in FIG. 11, Ang-(1-7) causes a dose-dependentreduction in Ang II-stimulated ERK activity in VSMCs, with maximalinhibition at 1 IM Ang-(1-7). Similarly, Ang-(1-7) at 1 μM shows maximalinhibition of serum-stimulated ERK1 and ERK2 activation in SK-LU-1 lungcancer cells (FIG. 12).

Ang-(1-7) also reduces ERK phosphorylation by platelet derived growthfactor (PDGF). Thus, 10 ng/mL PDGF increases ERK1 and ERK2 activities by16-fold and 26-fold in VSMCs. This stimulation is inhibited by almost50% by 1 μM Ang-(1-7). Also, as shown in FIG. 13, Ang-(1-7) inhibitsplatelet-derived growth factor (PDGF) or epidermal growth factor(EGF)-stimulated ³H-thymidine incorporation into human ZR-75-1 breastcancer cells by at least 50% (FIG. 13).

Thus, in an embodiment, Ang-(1-7) inhibits cell growth through areduction in the activity of mitogen-stimulated MAP kinases. Ang-(1-7)may also reduce MAP kinase activity by inhibiting the signaling pathwaysthat stimulate MAP kinase phosphorylation or by stimulating MAP kinasephosphatase activity.

Signal Transduction and Potential Molecular Mechanisms of Inhibition ofCancer Cell Growth and Proliferation by Ang-(1-7)

Cell division is a complex process that occurs with exquisite precisionsuch that each daughter cell receives the correct number of chromosomesand is capable of independent function. Cell cycle events are initiatedat the appropriate time, allowing for the completion of one phase beforethe next one is triggered. In an embodiment, angiotensin-(1-7) or otherangiotensin-(1-7) receptor agonists inhibit cancer cell growth and/orproliferation by increasing the expression of genes involved in tumorsuppression, apoptosis, and/or cell cycle inhibition. Alternatively, oradditionally, angiotensin-(1-7) or other angiotensin-(1-7) receptoragonists may inhibit cancer cell growth and/or proliferation bydecreasing the levels of known oncogenes, protein kinases, and/or cellcycle progression genes in the cancer. FIG. 14 shows a histogram of genetranscription increases and decreases when quiescent SK-LU-1 lung cancercells stimulated with 1% FBS for 2 h in the presence of 100 nM Ang-(1-7)as compared to SK-LU-1 lung cancer cells stimulated with 1% FBS for 2 hin the absence of Ang-(1-7).

Cyclin-dependent kinases (CDKs) are proteins involved in the control ofthe cell cycle. CDKs catalyze the covalent attachment of phosphate toprotein substrates, thereby altering the enzyme activity or proteinaffinity of the substrate. The regulation of the cellular concentrationsof CDKs leads to cyclical changes in the phosphorylation of keycomponents of the cell-cycle machinery, resulting in the initiation (orinhibition) of cell cycle events. CDKs are activated by binding toregulatory proteins called cyclins. Changes in CDK activity during thecell cycle are due primarily to the amount of cyclin proteins in thecell. In turn, cyclin concentrations are regulated at transcription andthrough proteolytic degradation of the cyclins at specific cell-cyclestages.

For example, two classes of CDK inhibitors-the p16/p19^(ARF) and p21families of proteins—bind to specific CDKs to prevent their interactionwith cyclins and thereby interfere with cyclin/CDK regulation of thecell cycle. Thus, p16^(INK4a) inhibits CDK4 and CDK6, resulting inhypophosphorylation of the retinoblastoma protein (Rb). When Rb isunder-phosphorylated, it binds to the transcriptional activator E2F toblock transcription and prevent progression through the cell cycle. Uponphosphorylation by CDK4 and 6, hyperphosphorylated Rb releases boundE2F, allowing it to increase the transcription of genes involved inprogression through the cell cycle (Lukas, J. et al., Nature, 1995,375:503-506;and Serraro, M. et al., Science, 1995, 267:249-252).

Cell cycle control of most cell types is also responsive toextracellular signals. Classic growth factors such as platelet-derivedgrowth factor (PDGF), epidermal growth factor (EGF), basic fibroblastgrowth factor (bFGF), or serum stimulate cell growth in vitro and invivo. Growth stimulation by these mitogens is mediated, at least inpart, through ras-Raf activation of mitogen-activated protein kinases(MAP kinases) to induce early response genes and increase transcription(Marrero, M. B. et al., J. Biol. Chem., 1997, 272:24684-24690; Molloy,C. J. et al., J. Biol. Chem, 1993, 268:7338-7345;and Pelech, S. L. andSanghera, J. S., Science, 1992, 257:1355-1356).

The Ras/Raf/MEK/ERK phosphorylation cascade is the prototypical cellularproliferation pathway documented in mammalian cells. Growth stimulationby many cytokines as well as by growth hormones also involves activationof the Janus kinase (JAK) family of cytosolic tyrosine kinases. Januskinases stimulate the phosphorylation of STAT (signal transducers andactivators of transcription) proteins, causing their translocation tothe nucleus and subsequent activation of transcription (Marrero, M. B.et al., J. Biol. Chem., 1997, 272:24684-24690;and Marrero, M. B. et al.,Nature, 1995, 375:247-250).

Thus, in an embodiment, application of a pharmaceutically effectiveamount of angiotensin-(1-7), or other angiotensin-(1-7) receptoragonists may inhibit cancer cell growth and/or proliferation bydecreasing the levels of known oncogenes, protein kinases, and/or cellcycle progression genes in cancer cells. In an embodiment, the genesshowing decreased expression comprise cell cycle entry regulator, ERK1,cell cycle progression 2 protein, p21/K-ras 2B oncogene, epithelial cellkinase, ser/thr kinase, MAP kinase kinase 5 (MEK5), beta catenin,tyrosine-protein kinase receptor tyro3 precursor (FIG. 14). As shown inFIG. 15, Ang-(1-7) decreases both MEK 5 mRNA and protein levels inSK-LU-1 lung cancer cells stimulated with serum. Thus, although MEK5protein levels increased immediately following mitogen stimulation, atboth 4 and 8 hours MEK5 mRNA and protein levels are reduced by treatmentwith Ang-(1-7).

Thus, in an embodiment, Ang-(1-7) agonists inhibit cancer cell growth byregulation of MAP kinase and JAK/STAT signaling pathways. This issupported by the findings that: (1) p21/ras mRNA and ERK1 mRNA andprotein are downregulated in serum-stimulated human SK-LU-1 lung cancercells following Ang-(1-7) treatment; (2) down-regulation of MEK5 mRNAand protein is observed in mitogen-stimulated human lung cells followingtreatment with Ang-(1-7); (3) Ang-(1-7) up-regulates the expression ofSOCS-3, a negative regulator of the JAK/STAT pathway in human lungcancer cells (FIG. 14) (Dey, B. R. et al., Biochem. Biophys. Res.Commun., 2000, 278:38-43;and Duhe, R. J. et al., Cell Biochem. Biophys.,2001, 34:17-59); and (4) Ang-(1-7) inhibits tumor growth in vivo.

Alternatively and/or additionally, Ang-(1-7) agonists inhibit cancercell growth and/or proliferation by increasing the levels of genesinvolved in tumor suppression and/or cell cycle inhibition. Preferably,the genes showing increased expression comprise pl6-INK, oncostatinM-specific beta subunit, PDCD2, EGF response factor 1, CASP4, RBQ-3,menin, checkpoint suppressor 1, SOCS-3, insulin-like growth factorbinding protein 2, B-myb or the fau tumor suppressor (FIG. 14).

Thus, under normal conditions, tissues maintain a balance between therates of cell proliferation and cell death. In contrast, tumor formationis a pathological state resulting from heightened cell division and areduced rate of apoptosis. Many cancer cells manifest an enhancedresistance to physiological stimuli that would ordinarily triggerapoptosis in normal cells. Substances that can stimulate apoptosis incancer cells may provide a novel mechanism for reducing cell number.

Caspase-3 is activated during apoptosis. In an embodiment, treatment ofcancer cells with Ang-(1-7) upregulates genes encoding the pro-apoptoticproteins BAD, BAK as well as apoptotic protease activating factor 1(FIG. 14) and increases the caspase-3 cleavage product ofpoly(ADP-ribose) polymerase (PARP) (FIG. 16) in mitogen-stimulatedcancer cells. An increase in the amount of caspase-3 cleavage productPARP by treatment with Ang-(1-7) indicates that Ang-(1-7) stimulatesapoptosis in lung cancer cells to thereby reduce cell growth.

Therapeutics

The invention contemplates methods of administration which are wellknown in the art. For example, in an embodiment, administration of thecompound is intravenous. In another embodiment, the method ofadministration is by a transdermal patch. Also, administration mayemploy a time-release capsule. In another embodiment, administration ofthe compound is intra-arterial. In yet another embodiment,administration of the compound is oral or as an aerosol. In anotherembodiment, administration of the compound is sublingual. In yet anotherembodiment, administration of the drug is transrectal, as by asuppository or the like.

Pharmaceutical formulations can be prepared by procedures known in theart. For example, the compounds can be formulated with commonexcipients, diluents, or carriers, and formed into tablets, capsules,suspensions, powders, and the like. Examples of excipients, diluents,and carriers, that are suitable for such formulations include thefollowing: fillers and extenders such as starch, sugars, mannitol, andsilicic derivates; binding agents such as carboxymethyl cellulose andother cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone; moisturizing agents such as glycerol; disintegrating agentssuch as agar, calcium carbonate, and sodium bicarbonate; agents forretarding dissolution such as paraffin; resorption accelerators such asquaternary ammonium compounds; surface active agents such as cetylalcohol, glycerol monostearate; adsorptive carriers such as kaolin andbentonite; and lubricants such as talc, calcium and magnesium stearate,and solid polyethyl glycols.

The compounds can also be formulated as elixirs or solutions forconvenient oral administration or as solutions appropriate forparenteral administration, for instance by intramuscular, subcutaneousor intravenous routes. Additionally, the compounds are well suited toformulation as sustained release dosage forms and the like. Theformulations can be so constituted that they release the activeingredient only or preferably in a particular part of the intestinaltract, possibly over a period of time. The coatings, envelopes, andprotective matrices may be made, for example, from polymeric substancesor waxes.

The therapeutic efficacy of exogenous compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals using procedures known in the art. The dose ratio between toxicand therapeutic effects is the therapeutic index and may be expressed asLD₅₀/ED₅₀, wherein LD₅₀ is understood to represent the dose which istoxic to 50% of the subjects and ED₅₀ is understood to represent thedose which is effective in 50% of the subjects. Generally, compoundswhich exhibit large therapeutic indices are preferred. Administration ofthe compound may be hourly, daily, weekly, monthly, yearly or a singleevent.

In an embodiment, the dose of Ang-(1-7) agonist required for inhibitionof cancer cells comprises levels of angiotensin-(1-7) agonist that areused pharmacologically in animals and humans. Also preferably, the doseof angiotensin-(1-7) receptor agonist results in a local concentrationof angiotensin-(1-7) agonist at the tumor which ranges from 0.005 nM to10 μM, and more preferably, from 0.05 nM to 1 μM, or even morepreferably, from 1 nM to 100 nM. Also, the ability of Ang-(1-7) agoniststo inhibit tumor growth may a function of cell division and the lengthof the cell cycle. Thus, application of the Ang-(1-7) agonist may behourly, daily, or over the course of weeks. Thus, preferably, theeffective amount of the Ang-(1-7) agonist comprises from about 1 ng/kgbody weight to about 100 mg/kg body weight. More preferably, theeffective amount of the Ang-(1-7) agonist comprises from about 1 μg/kgbody weight to about 50 mg/kg body weight. Even more preferably, theeffective amount of the Ang-(1-7) agonist compound comprises from about10 μg/kg body weight to about 10 mg/kg body weight. Alternatively, acontinuous level of Ang-(1-7) agonist ranging from about 0.05-1,000μg/kg/hour, or more preferably, 0.5-250 μg/kg/hr, or even morepreferably 5-50 μg/kg/hour may be employed. The actual effective amountwill be established by dose/response assays using methods standard inthe art. Thus, as is known to those in the art, the effective amountwill depend on bioavailability, bioactivity, and biodegradability of thecompound.

EXAMPLES

The present invention will be further understood by reference to thefollowing non-limiting examples.

Example 1 Materials and Methods

A. Angiotensin Receptor Pentides and Non-peptide Compounds

All angiotensin peptides (natural and modified) were obtained fromBachem, Torrance, Calif. AT₁ antagonists Losartan and L158,809 wereobtained from Merck & Co., Inc., Rahway, N.J. The AT₂ antagonistPD123177 was obtained from Parke-Davis Pharmaceutical Research, AnnArbor, Mich.

B. Rat Vascular Smooth Muscle Cells (VSMCs) and Human Lung Cancer Cells

Vascular smooth muscle cells were isolated form 12-14 week oldSprague-Dawley rats by explant culture (Freeman, E. J. et al.,Hypertension, 1996, 28:104-108). Human lung and breast cancer cell lineswere obtained from American Type Tissue Culture (ATTC) and includedcells of the SK-LU-1 and A549 cell lines (both of which are derived fromadenocarcinomas) as well as SK-MES-1 cells (derived from non-small celllung tumors) and ZR-75-1 breast cancer cells.

Cells were grown in DMEM with 10% fetal bovine serum (FBS), 100 μg/mLpenicillin and 100 units/mL streptomycin in a humidified 37° C.incubator gassed with 5% CO₂ and 95% room air. Cells were grown tosubconfluence in either 24-well cluster plates or 100 mm dishes and madequiescent by treatment for 48 h with serum-depleted growth media, priorto the experiments outlined below to measure cell growth (³H-thymidineincorporation), cell signaling or apoptosis.

C. Analysis of ³H-thymidine Incorporation

To measure ³H-thymidine incorporation, quiescent cells were incubatedwith serum and angiotensin peptides for 24 h at 37° C. ³H-thymidine(0.25 μCi/well) was added and the cells incubated for an additional 4 hto incorporate the radiolabeled nucleotide. Subsequently, the cellmonolayer was washed with cold phosphate-buffered saline (PBS; 50 mMNaPO₄, 120 mM NaCl, pH=7.2). The adherent cells were precipitated withcold 10% TCA (4° C. for 30 min) and dissolved in 0.2% SDS in 0.1 N NaOH.Incorporated ³H-thymidine was determined by liquid scintillationspectrometry, as previously described (Freeman, E. J. et al.,Hypertension, 1996, 28:104-108). Growth inhibition was defined as areduction in the amount of ³H-thymidine incorporation as compared to themitogen-stimulated controls.

To study the receptor specificity of the effect, cell monolayers werepreincubated with 1 μM of the AT₁ antagonist Losartan (or L158,809), theAT₂ antagonist PD 123319, the non-selective angiotensin peptideantagonist [Sar¹-Thr⁸]-Ang II, or the Ang-(1-7)-selective antagonist[D-Ala⁷]-Ang-(1-7), followed by treatment with various doses of mitogensand Ang-(1-7). Quiescent cells were stimulated with increasingconcentrations of Ang-(1-7) and/or antagonists for 24 h. During anadditional 4 h, cell monolayers were pulsed with ³H-thymidine (0.25μCi/well) and harvested. Cells treated with mitogen and antagonists inthe absence of Ang-(1-7) were used as the controls, to detect any effectof the antagonists alone.

D. Statistics

For all experiments, cells were used from at least three differentpassage numbers of each cell type. Values are expressed as mean±standarderror of the mean. Statistical significance of differences was evaluatedby one way analysis of variance with p values corrected by Dunnett'spost test, using the statistics package Instat (GraphPad). The criterionfor statistical significance was set at p<0.05.

E. RT-PCR and Western Analysis

For RT-PCR, total RNA was isolated using the Atlas Pure Total RNALabeling System (Clontech Laboratories, Inc). The RNA concentration wasquantified by UV spectroscopy and any degradation assessed by ethidiumbromide staining intensity of 28S and 18S ribosomal RNA followingagarose gel electrophoresis. The isolated RNA was incubated with DNaseto eliminate any residual DNA, and approximately 250 ng of total RNA persample incubated with or without AMV reverse transcriptase in a mixturecontaining deoxynucleotides, random hexamers, and RNase inhibitor inreverse transcriptase buffer. The mixture was heated for 5 min at 95° C.to terminate the reaction. For amplification of the resulting cDNA, 1μmol/L gene-specific primers, 0.2 mmol/L deoxynucleotides, 5μCi³²P-dCTP, 1.5 mmol/L MgCl₂, and 1.5 U Taq DNA polymerase was added to3 μL of the RNA sample in a final volume of 50 μL. As an internalstandard, primers specific for the gene encoding Elongation Factor 1αwere added. Following PCR, the amplification products were separated bypolyacrylamide gel electrophoresis, visualized by autoradiography, andanalyzed using the MCID imaging system.

For Western blot analysis, quiescent cells treated with Ang-(1-7) andserum for various periods of time, between 2 and 24 h, were solubilizedin SDS and protein content analyzed using the modified Lowry method(Lowry, O. H., et al., J. Biol. Chem., 1951, 193:265-275). Proteins wereseparated electrophoretically on SDS polyacrylamide gels, transferred topolyvinyl membranes, and incubated with primary antibodies to proteinsof interest. Appropriate horseradish peroxidase (HRP)-conjugated secondantibodies were added and immunoreactive products visualized using theenhanced chemoluminescence reagents from Amersham. The density of eachimmunoreactive product was quantified using the MCID imaging system.Antibodies to proteins that participate in cell signaling, apopotosis,and regulation of the cell cycle are commercially available from avariety of sources.

F. Measurement of MAP Kinases

Quiescent lung cancer cells were incubated with increasingconcentrations of Ang-(1-7) [from 10⁻⁹ to 10⁻⁶ M] for 10 min at roomtemperature. Reactions were terminated with Triton lysis buffer (50 mMTris-HCI, pH 7.4, 1% Triton X-100, 100 mM NaCl, 5 mM EDTA, 50 mM NaF,0.6 μM leupeptin, 0.01 mM Na₃VO₄ and 0.1 mM PMSF) and proteinconcentrations determined (Lowry, O.H., et al., J. Biol. Chem., 1951,193:265-275). Proteins were separated by SDS polyacrylamide gelelectrophoresis and transferred to polyvinyl membranes. The activationand autophosphorylation of ERK1 and ERK2 was determined using antibodiesspecific for the phosphorylated kinases (using antibodies from CellSignaling Technologies). The immunoreactive product were visualized byenhanced chemiluminescence (ECL, Amersham) and quantified bydensitometry, using the MCID image analysis system. The phospho-MAPkinase antibodies only recognize the catalytically activated andphosphorylated forms of MAP kinase (both ERK1 and ERK2 MAP kinase). Theblots were also probed with antibodies to ERK1/2, to control for proteinloading.

Example 2 Inhibition of Vascular Growth by Ang-(1-7) In Vitro

These experiments support earlier indications (Freeman, E. J. et al.,Hypertension, 1996, 28:104-108;and Tallant, E. A. et al., Hypertension,1999, 34:950-957) that Ang-(1-7) inhibits the growth of culturedvascular smooth muscle cells (VSMCs). Incorporation of ³H-thymidine intoVSMCs obtained from rat thoracic aorta was significantly increased byincubation with fetal bovine serum (FBS), platelet-derived growth factor(PDGF), or Ang II. Following a 48 hr treatment with 1 μM Ang-(1-7), theincorporation of ³H-thymidine in response to 1% FBS, 10 ng/mL PDGF and100 nM Ang II was markedly attenuated (to 66.4, 84.3, and 75.8% ofmitogen-stimulated activity, respectively). The reduction inserum-stimulated thymidine incorporation by Ang-(1-7) wasdose-dependent, with a peak effect at a dose of 1 tM and an IC₅₀ of 115nM (FIG. 1). Maximal inhibition by 1 μM Ang-(1-7) was approximately 60%of control in the presence or absence of FBS, which is similar to thegrowth inhibition previously reported for atrial natriuretic factor(ANF) (Appel, R. G., Am. J. Physiol., 1990, 259:E312-E318).

Total cell number in response to treatment with Ang-(1-7) was alsodetermined using a Coulter counter. The number of cells per wellincreased to 142% of basal following treatment with 1% serum. Treatmentof serum-stimulated cells with 1 μM Ang-(1-7) significantly reduced thenumber of cells per well (to 109% of basal). By comparison, Ang IIincreased the number of cells per well to 145% of basal values andcaused a dose-dependent stimulation of ³H-thymidine incorporation intoVSMCs, as shown in FIG. 1. Thus, Ang-(1-7) inhibits mitogen-stimulatedVSMC growth and opposes the proliferative effects of Ang II.

Example 3 The Effects of Ang-(1-7) Are Mediated Via a Specific Ang-(1-7)Receptor

It is documented that the mitogenic effect of Ang II is mediated by theAT₁ angiotensin receptor and that stimulation of vascular AT₂ receptorsinhibits growth. However, attenuation of (fetal bovine) serum-stimulatedthymidine incorporation (FBS) by Ang-(1-7) (+A7) was unaffected byantagonists selective for AT₁ (L158,809) or AT₂ (PD 123177) receptors(FIG. 2). In contrast, a 10-fold molar excess of the non-selectiveangiotensin receptor antagonist ([Sar¹-Thr⁸]-Ang II; Sarthran)completely blocked growth inhibition by Ang-(1-7), indicating that theeffect of the heptapeptide was a result of the activation of anangiotensin receptor pharmacologically distinct from either AT₁ or AT₂receptors. [D-Ala⁷]-Ang-(1-7) also blocked the growth inhibitoryresponse to Ang-(1-7). The substitution of D-alanine for proline inAng-(1-7) results in a molecule that has no agonistic activity, does notcompete at AT₁ or AT₂ receptors, and selectively blocks hemodynamic andrenal responses to Ang-(1-7) (Santos, R. A. S. et al., Brain Res. Bull.,1994, 35:293-298). These data indicate that Ang-(1-7) inhibits VSMCgrowth through activation of a non-AT₁, non-AT₂ receptor that issensitive to [Sar¹-Thr⁸]-Ang II and [D-Ala⁷]-Ang-(1-7), the AT₍₁₋₇₎receptor.

Example 4 In Vivo Studies of Ang-(1-7) Reduction of Vascular Cell Growth

To study the role of the peptide in vivo, the effect of Ang-(1-7) onvascular growth stimulated by balloon catheter injury to the rat carotidartery was determined. Intravenous infusion of Ang-(1-7) with achronically implanted minipump [24 μg/kg/h, 5 μL/h, 12 days] increasedthe plasma Ang-(1-7) concentration to 131.4±39.7 pM (n=5) from 42.2±10.7pM (n=8) in carotid artery-injured rats infused with saline. Plasmaconcentrations of Ang II, blood pressure and heart rate were similar inrats infused with Ang-(1-7) or saline. Morphometric analysis of carotidartery cross-sections indicated that Ang-(1-7) infusion significantlyreduced the neointimal area compared to rats infused with saline(0.10±0.009 mm² vs. 0.066±0.012 mm², respectively; p<0.05) but had noeffect on the medial area of the injured or the contralateral uninjuredartery as compared to saline controls (FIGS. 3 and 4) (Strawn, W. B. etal., Hypertension, 1999, 33:207-211and Tallant, E. A. et al.,Hypertension, 1999, 34:950-957). Thus, Ang-(1-7) inhibits vasculargrowth in vivo. The antiproliferative effect of Ang-(1-7) in preventingneointimal growth is of clinical importance as vascular re-stenosismediated by a proliferative response of smooth muscle in blood vesselsis a complication of surgical procedures that use stents to preventvessel occlusion following crushing of an atherosclerotic plaque(angioplasty).

The concentrations of Ang-(1-7) shown to be effective in reducingvascular growth in response to injury are similar to plasma levels ofAng-(1-7) in rats following balloon catheter injury and treated with theACE inhibitor lisinopril (20 mg/kg/day for 14 days). Inlisinopril-treated rats, plasma levels of Ang-(1-7) were elevated2.3-fold (from 42.3±6.7 pM in saline treated animals (n=10) to 99.1±6.7pM (n=10)). In these same animals, the cross-sectional area of theneointima was reduced to 0.09±0.01 mm² as compared to 0.12±0.02 mm² insaline-treated controls (p<0.05). Thus, exogenous Ang-(1-7) infusion ortreatment with the ACE inhibitor lisinopril to increase Ang-(1-7)reduced neointimal formation after vascular injury at concentrations ofthe peptide only two-fold higher than in saline-treated rats.

Example 5 Inhibition of Human Cancer Cell Growth by Ang-(1-7)

These experiments show that Ang-(1-7) reduces the growth of lung andbreast cancer cells. Ang-(1-7) inhibited serum-stimulated ³H-thymidineincorporation into human lung cancer cells of the A549, SK-MES-1, andSK-LU-1 cell lines and the ZR-75-1 breast cancer cell line. Theattenuation of human lung adenocarcinoma SK-LU-1 cell growth wasdependent on the dose of Ang-(1-7) with a maximal reduction of 33.8±5.3%of serum-stimulated growth and an IC₅₀ of 0.05 nM, as shown in FIG. 5.Ang-(1-7) also attenuated mitogen-stimulated growth of human lungadenocarcinoma A549 cells (maximal inhibition of 41.3±10.9%, IC₅₀=0.11nM) as well as non-small cell lung cancer SK-MES-1 cells (maximalinhibition of 40.9±2.9%, IC₅₀=0.4 nM) and breast cancer ZR-75-1 cells(maximal inhibition of 37.2±6.1; IC₅₀=0.02 nM). Thus, Ang-(1-7) reduceshuman lung and breast cancer cell growth in a dose-dependent manner withIC₅₀ levels similar to circulating levels of Ang-(1-7) measured aftertreatment of rats with the ACE inhibitor lisinopril (Campbell, D. J. etal., Hypertension, 1993, 22:513-522;and Kohara, K. et al., Circulation,1991, 84 (supp. II):662).

The inhibition of growth by Ang-(1-7) was also dependent upon the timeof treatment with Ang-(1-7). The incorporation of ³H-thymidine intoSK-LU-1, A549, and SK-MES-1 lung cancer cells and ZR-75-1 breast cancercells stimulated to grow by the inclusion of 1% FBS was progressivelyreduced by daily addition of 100 nM Ang-(1-7), as shown in FIG. 6.Ang-(1-7) was renewed daily due to the endogenous degradation of thepeptide (Chappell, M. C. et al., Hypertension, 1998, 31:362-367). Theseresults suggest that Ang-(1-7), an endogenous peptide, inhibits themitogen-stimulated growth of lung cancer cells.

Inhibition of the serum-stimulated growth of SK-LU-1 human lung cancercells by Ang-(1-7) was blocked by the Ang-(1-7) selective antagonist[D-Ala⁷]-Ang-(1-7), while neither AT₁ nor AT₂ angiotensin receptorantagonists Losartan and PD123177, respectively, were effective (FIG.7). This suggests that the anti-proliferative effect of Ang-(1-7) inlung cancer cells is mediated by a novel AT₍₁₋₇₎ receptor.

Also the effects are specific to Ang-(1-7), and are not exhibited byother angiotensin peptides. Thus, neither Ang I, Ang-(2-8) or Ang III,Ang-(3-8) or Ang IV, Ang-(3-7), nor Ang II mimicked the growth inhibitoreffects of Ang-(1-7), as shown in FIG. 8. These results suggest that theanti-proliferative effect of Ang-(1-7) is mediated by a novel Ang-(1-7)receptor and may represent a new therapeutic treatment for thesecancers.

Example 6 Inhibition of Tumor Growth by Ang-(1-7)

To determine whether Ang-(1-7) inhibits tumor growth in vivo, athymicmice were inoculated subcutaneously in the lower flank withapproximately 1.5×10⁷ cells of the ZR-75-1 breast cancer cell line.Tumor volumes were measured by caliper two times per week and calculatedusing the formula for a semiellipsoid. After 40 days, the mice hadtumors approximately 175 mm³ in size and were randomized for treatment.Primed osmotic mini-pumps (delivery rate of 0.25 μL/hr) were implantedonto the backs of the mice with the control group receiving continuousintravenous infusion of saline (6 μL/24 hr) and the experimental groupreceiving Ang-(1-7) (24 μg/kg/hr) for 28 days. The dose of Ang-(1-7) wasbased on previous studies with rats, which indicated that this dose wastolerated with no change in weight, blood pressure, or heart rate andresulted in a 2 to 3-fold elevation in circulating Ang-(1-7) (Strawn, W.B. et al., Hypertension, 1999, 33:207-211. As shown in FIG. 9, anapproximate 40% reduction in tumor volume was observed in mice treatedwith Ang-(1-7) for 4 weeks, while the tumor size doubled in thesaline-treated animals, as compared to tumor size prior to treatment.These results show that Ang-(1-7) inhibits breast tumor growth in vivoand that Ang-(1-7) is an effective therapeutic agent in vivo.

Example 7 Mechanism of Growth Inhibition by Ang-(1-7)

It was found that Ang-(1-7) stimulates prostacyclin (PGI₂) release fromVSMCs isolated from Sprague-Dawley rat aortas, measured as the releaseof the stable metabolite of prostacyclin, 6-keto-PGF_(1α). Ang-(1-7)caused a dose-dependent release of prostacyclin, with maximal release of177.9±25.2% above basal release at 100 nM Ang-(1-7). Since prostacyclininhibits VSMC growth, these results suggest that Ang-(1-7) attenuatesvascular growth through the production and release of prostacyclin.

Prostacyclin is produced by the cyclooxygenase-mediated conversion ofarachidonic acid into PGG₂/PGH₂, which is subsequently processed byprostacyclin synthase into prostacyclin. Interestingly, thecyclooxygenase inhibitor indomethacin (IND, 10 μM) effectively blockedthe growth inhibition mediated by Ang-(1-7) (97.4±3.6% of control, n=4,p<0.05) compared to the decrease of serum-stimulated ³H-thymidineincorporation by Ang-(1-7) in the absence of indomethacin (79.1±5.1% oftotal, n=5, p<0.05). Since neither a lipoxygenase inhibitor nor thecytochrome P450 inhibitor 1 7-octadecynoic acid had any effect on growthinhibition by the heptapeptide, these results show that Ang-(1-7)inhibits VSMC growth through the production of a metabolite of thecyclooxygenase pathway which may be prostacyclin.

The addition of prostacyclin or carbacyclin (5 μM; Calbiochem, La JollaCalif.) (a stable analogs of prostacyclin) to VSMCs activates adenylatecyclase resulting in an elevation in the cellular levels of cAMP.Ang-(1-7), at a concentration of 1 μM, caused a significant increase inthe cellular levels of cAMP, to 131.9±9.7% of basal (n=3, p<0.05), inthe presence of 1 mM isobutylmethyl xanthine (IBMX), a cyclic nucleotidephosphodiesterase inhibitor. cAMP activates a cAMP-dependent proteinkinase, protein kinase A. As shown in FIG. 10, the reduction inserum-stimulated ³H-thymidine incorporation by Ang-(1-7) or carbacyclinwas completely blocked by pretreatment with the protein kinase Ainhibitor (PKAI) Rp-adenosine-3′,5′-cyclic monphospho-phorothioatetriethylamine salt (Rp-cAMPS) (Calbiochem, La Jolla Calif.). Theseresults suggest that Ang-(1-7) is directly coupled to the Gs protein toactivate adenylate cyclase and elevate cellular cAMP production.Alternatively, Ang-(1-7) may stimulate the production of prostacyclinwhich binds to prostacyclin receptors coupled to adenylate cyclase andthe synthesis of cAMP. Collectively, these results suggest thatAng-(1-7) causes an increase in the cellular levels of cAMP whichstimulates the cAMP-dependent protein kinase to inhibit growth.

Ang-(1-7) may inhibit cell growth by preventing the phosphorylation andactivation of MAP kinases in response to mitogen stimulation. Forexample, compounds that increase the intracellular concentration of cAMPhave been shown to reduce MAP kinase activity in VSMCs and fibroblastsand inhibit mitogen-stimulated growth in VSMCs (Cook, S. J. andMcCormick, F., Science, 1993, 262:1069-1072;and Wu, J. et al., Science,1993, 262;1065-1068). In addition, classic growth factors, such as PDGF,epidermal growth factor, and basic fibroblast growth factor stimulateVSMC growth in vitro and in vivo. Growth stimulation by these mitogensas well as by Ang II is mediated, at least in part, through activationof MAP kinases to induce early response genes and increasetranscription.

The activity of the MAP kinases ERK1 and ERK2 in VSMCs was measuredusing phospho-specific antibodies that only recognize the activatedprotein kinases. Ang II caused a dose-dependent increase in both ERK1and ERK2 phosphorylation (37- and 166-fold increase over basal), withmaximal stimulation by 1 μM Ang II. Incubation of VSMCs withconcentrations of Ang-(1-7) up to 1 μM had no effect on ERK1 or ERK2phosphorylation. However, pre-incubation with increasing concentrationsof Ang-(1-7) caused a dose-dependent reduction in Ang II-stimulated ERKactivity, with maximal inhibition at 1 μM Ang-(1-7). One micromolarAng-(1-7) reduced 100 nM Ang II-stimulated ERK1 and ERK2 activation by42.3±6.2% and 41.2±4.2%, p<0.01, respectively, as shown in FIG. 11.

Ang-(1-7) also reduced ERK phosphorylation by 10 ng/mL PDGF in VSMCs,which increased ERK1 and ERK2 activities by 16-fold and 26-fold overbasal, respectively. It was found that 1 μM Ang-(1-7) decreasedPDGF-stimulated ERK1 and ERK2 activities by 43.6±8.6% and 38.7±11.4%,p<0.05, respectively.

To begin to address the molecular mechanisms of Ang-(1-7) inhibition ofthe growth of cancer cells, MAP kinase activity was measured inquiescent SK-LU-1 cells stimulated with 1% FBS, in the presence andabsence of increasing concentrations of Ang-(1-7). As shown in FIG. 12,Ang-(1-7) caused a dose-dependent decrease in the amount ofserum-stimulated ERK1 and ERK2 activities. These results suggest thatAng-(1-7) either inhibits the kinase which phosphorylates and activatesERK1 and ERK2, a MAP kinase kinase, or stimulates the activity of a MAPkinase phosphatase, either of which would result in a decrease in activeMAP kinase.

Ang-(1-7) also inhibited platelet-derived growth factor (PDGF)- orepidermal growth factor (EGF)-stimulated ³H-thymidine incorporation intohuman breast cancer cells of the ZR-75-1 cell line, as shown in FIG. 13.For these experiments, semi-confluent cell monolayers were madequiescent by a 48-h incubation in serum-free media, followed by a 28-htreatment period with increasing concentrations of Ang-(1-7) in thepresence of either 2.5 ng/mL PDGF or 100 ng/ml EGF. It was found thatAng-(1-7) reduces mitogen-stimulated human breast cancer cell growth ina dose-dependent manner.

The results show that Ang-(1-7) attenuates MAP kinase activation byeither Ang II, serum, or growth factors PDGF or EGF, and that Ang-(1-7)can inhibit cell growth through a reduction in the activity ofmitogen-stimulated MAP kinases. Thus, Ang-(¹-7) may reduce MAP kinaseactivity by inhibiting the signaling pathways that stimulate MAP kinasephosphorylation or by stimulating MAP kinase phosphatase activity.

Example 8 Mechanisms of Inhibition of Cancer Cell Growth by Ang-(1-7)

To further assess transcriptional regulation involved in the inhibitionof cancer cell growth and proliferation by Ang-(1-7), total RNA isolatedfrom SK-LU-1 cells treated with 1% serum in the presence and absence of100 nM Ang-(1-7) was analyzed using gene array hybridization. Cells wereincubated for 2 or 8 h, and total RNA was isolated using the Atlas PureTotal RNA Labeling System (Clontech Laboratories, Inc). The RNAconcentration was quantified by UV spectroscopy and any degradation wasassessed by ethidium bromide staining intensity of 28S and 18S ribosomalRNA following agarose gel electrophoresis. RNA isolated from sevendifferent cell passages was pooled prior to gene array analysis toaccount for individual variability in gene regulation. RadiolabeledcDNA, prepared from the pooled RNAs using the Atlas system, wasincubated with DNase to degrade any residual DNA and then hybridized tothe Human Cancer 1.2 Atlas CDNA Expression Array (ClonetechLaboratories, Inc). This gene array set contains 1,176 characterizedhuman cDNAs on positively-charged nylon membranes. The resultanthybridization signals, visualized by phosphorimage analysis, werequantified using the computerized MCID imaging system with gene arrayanalysis software to identify potential gene products which areup-regulated or down-regulated in response to Ang-(1-7) stimulation.

FIG. 14 shows some of the results obtained by gene array hybridization.A number of genes involved in tumor suppression, apoptosis, and cellcycle inhibition were upregulated in SK-LU-1 cells treated withAng-(1-7), including the tumor suppressors p16^(INK4a) and menin andgenes encoding the proapoptotic proteins BAD and BAK as well asapoptotic protease activating factor 1. In contrast, several oncogenes,protein kinase and cell cycle progression genes were downregulated. Forexample, MAP kinase kinase 5 (MEK5), ERK1, and p21/K-ras 2B werereduced, suggesting that Ang-(1-7) may also chronically reduce theRas/Raf/MEK/MAP kinase signaling cascade. These results suggest a numberof signaling pathways that may be involved in the Ang-(1-7)-mediatedreduction of cell proliferation observed in the lung cancer cells.Several candidate genes were selected for verification by RT-PCR andWestern analysis.

The gene array hybridization results indicated that MAP kinase kinase 5(MEK5) was downregulated in response to Ang-(¹-7). Thus, MEK5 expressionin SK-LU-1 lung cancer cells in response to Ang-(1-7) was measured byRT-PCR and Western analysis. Quiescent SK-LU-1 cells were stimulatedwith 1% serum in the presence and absence of 10 nM Ang-(1-7). RNA wasisolated using Trizol and whole cell lysates were isolated at 2, 4, 8and 24 h following treatment. As shown in FIG. 15, MEK5 mRNA and proteinwere reduced 4 and 8 h following treatment with Ang-(1-7). The resultsof these experiments indicate that the cellular concentrations of MEK5,a protein involved in MAP kinase signaling and cell growth, are reducedin human lung cancer cells following treatment with Ang-(1-7). In FIG.15, intensities of RT-PCR products and protein bands were determined byimage analysis. MEK5 protein levels increased immediately after mitogenstimulation. Still, at both 4 and 8 hours, MEK5 mRNA and protein levelsare reduced by treatment with Ang-(1-7).

The gene array hybridization results also indicated that mRNAs encodingproteins that stimulate or participate in apoptosis (BAD, BAK, and APAF)are upregulated by Ang-(1-7) in mitogen-stimulated SK-LU-1 cells.Stimulation of apoptosis by Ang-(1-7) was measured by generation of thecaspase-3 cleavage product poly(ADP-ribose) polymerase (PARP), todetermine whether Ang-(1-7) stimulates apoptosis. Since casepase-3 isactivated during apoptosis, an increase in the generation of itscleavage product (PARP) is a measure of apoptosis. Cleaved PARP wasmeasured using an anti-cleaved product-specific antibody inserum-stimulated SK-LU-1 cells treated for either 2, 4, or 8 hours with10 nM Ang-(1-7). As shown in FIG. 16, an increase in the amount ofcleaved PARP was visualized following a 4 to 8 hour treatment withAng-(1-7), suggesting that Ang-(1-7) stimulates apoptosis. These resultssuggest that, in human lung cancer cells, Ang-(1-7) stimulates apoptosisto reduce cell growth.

The regulation of cell growth is a key element in the normal maintenanceof healthy tissue. A delicate balance exists between the proliferativeand anti-proliferative factors controlling cell growth. Theidentification of the molecular mechanisms regulating cell growth isvital to understanding tumor formation, and the development ofanti-cancer therapeutices. In an embodiment, the present inventionrecognizes that Ang-(1-7), a peptide hormone present in the circulation,causes a marked decrease in cell proliferation of vascular cells as wellas cancer cell growth in vitro and in vivo. Ang-(1-7) is present in thecirculation at concentrations similar to the vasoconstrictor peptidehormone Ang II, and is generated from the precursor Ang I or Ang II bytissue peptidases and ACE inhibitors. Thus, in an embodiment, thepresent invention describes the use of a pharmaceutically effectiveamount of angiotensin-(1-7) or an angiotensin-(1-7) receptor agonist asa means to prevent tumor formation, or inhibit tumor growth in anindividual. The invention has been described in detail with particularreference to preferred embodiments thereof, but it will be understoodthat variations and modifications can be effected within the spirit andscope of the invention.

1. A composition for inhibition of at least one of cancer cell growth orproliferation comprising a pharmaceutically effective amount of anagonist for the angiotensin-(1-7) receptor in a pharmaceuticallyacceptable carrier formulated for delivery of the agonist to theangiotensin-(1-7) receptor on the cancer cells, wherein theangiotensin-(1-7) receptor agonist comprises angiotensin-(1-7) peptidehaving the amino acid sequence set forth in SEQ ID NO: 1, and wherein apharmaceutically effective amount of angiotensin-(1-7) receptor agonistcomprises an amount determined to be sufficient to inhibit cancer cellgrowth or proliferation by binding to the angiotensin-(1-7) receptor onthe cancer cell, and ranges from 0.01 nM to 100 nM angiotensin-(1-7). 2.The composition of claim 1, wherein the cancer cells comprise breastcancer, brain cancer, colon cancer, or lung cancer cells.
 3. Thecomposition of claim 1, wherein the cancer cells are in a human subject.4. The composition of claim 1, wherein the angiotensin-(1-7) receptoragonist is modified to increase its chemical stability in vivo.
 5. Thecomposition of claim 1, wherein the pharmaceutically effective amount ofthe angiotensin-(1-7) receptor agonist increases cellular prostacyclinsin the cancer cells.
 6. The composition of claim 1, wherein thepharmaceutically effective amount of the angiotensin-(1-7) receptoragonist increases cellular cAMP in the cancer cells.
 7. The compositionof claim 1, wherein the pharmaceutically effective amount of theangiotensin-(1-7) receptor agonist increases the expression of at leastone gene involved in tumor suppression, apoptosis, and/or cell cycleinhibition in the cancer cells.
 8. The composition of claim 7, whereinthe gene showing increased expression comprises a gene encoding BAD,oncostatin M-specific beta subunit, PDCD2, EGF response factor 1, CASP4,RBQ-3, p16-INK, menin, checkpoint suppressor 1, BAK, apoptotic proteaseactivating factor-1, SOCS-3, insulin-like growth factor binding protein2, B-myb or the fau tumor suppressor.
 9. The composition of claim 1,wherein the pharmaceutically effective amount of the angiotensin-(1-7)receptor agonist decreases the levels of expression of at least oneknown oncogene, protein kinase gene, and/or cell cycle progression genein the cancer cells.
 10. The composition of claim 9, wherein the geneshowing decreased expression comprises a gene encoding a cell cycleentry regulator, ERK1, cell cycle progression 2 protein, p21/K-ras 2Boncogene, epithelial cell kinase, ser/thr kinase, MAP kinase kinase 5(MIEK5), beta catenin, tyrosine-protein kinase receptor tyro3 precursor,protein phosphatase 2A B56-alpha, cyclin-dependent kinase regulatorysubunit (CDC28), cell division protein kinase 6 (CDK6), c-myc oncogene,ERBB-3 receptor protein tyrosine kinase, A-kinase anchoring protein, orrho C.
 11. The composition of claim 1, wherein the amount ofangiotensin-(1-7) receptor agonist ranges from 0.1 nM to 100 nM.
 12. Thecomposition of claim 1, wherein the amount of angiotensin-(1-7) receptoragonist ranges from 1 nM to 100 nM.
 13. The composition of claim 1,wherein the amount of angiotensin-(1-7) receptor agonist ranges from 10nM to 100 nM.
 14. A kit for inhibiting at least one of cancer cellgrowth or proliferation in an individual comprising: (a) at least onecontainer comprising a pharmaceutically effective amount of a functionalagonist for the angiotensin-(1-7) receptor, wherein the agonist for theangiotensin-(1-7) receptor comprises angiotensin-(1-7) peptide havingthe sequence set forth in SEQ ID NO: 1, and wherein a pharmaceuticallyeffective amount comprises an amount of angiotensin-(1-7) receptoragonist determined to be sufficient to inhibit cancer cell growth orproliferation by binding to an angiotensin-(1-7) receptor on the cancercell and ranges from 0.01 nM to 100 nM angiotensin-(1-7); (b) apharmaceutically acceptable carrier formulated for delivery of theagonist to the angiotensin-(1-7) receptor on the cancer cell; and (c)instructions for use.
 15. The kit of claim 14, wherein the cancer cellscomprise, breast cancer, brain cancer, colon cancer, or lung cancercells.
 16. The kit of claim 14, wherein the angiotensin-(1-7) receptoragonist is modified to increase its chemical stability in vivo.
 17. Thekit of claim 14, wherein the amount of angiotensin-(1-7) receptoragonist ranges from 0.1 nM to 100 nM.
 18. The kit of claim 14, whereinthe amount of angiotensin-(1-7) receptor agonist ranges from 1 nM to 100nM.
 19. The kit of claim 14, wherein the amount of angiotensin-(1-7)receptor agonist ranges from 10 nM to 100 nM.